Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myeloid diseases characterized by excessive myeloid proliferation and reduced numbers of red cells and platelets. Thus, they have features of both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). These MDS/MPN disorders carry a very poor prognosis, with most patients dying within 2 years of diagnosis. Somatic NRAS and KRAS mutations are common in MDS/MPN, and mice that express an activated KrasD12 allele develop a similar disease. This is characterized by profound myelomonocytic proliferation, severe anemia, early death and complete penetrance. Pathogenic bone marrow progenitors in KrasD12 mice demonstrate: (1) biased differentiation that favors the granulocyte/macrophage (GM) lineage at the expense of early megakaryocyte- erythroid (MegE) progenitors, (2) cytokine independent growth and (3) cell-intrinsic hypersensitivity to serum and the cytokine GM-CSF in committed GM progenitors. Despite intensive study of Ras signaling, the basic mechanisms by which Ras activation causes excessive myeloid cell production and anemia in MDS/MPN are unknown. Inhibition of the Raf/MEK/ERK cascade downstream of Ras was recently found to extend the lifespan of KrasD12 mice and correct progenitor abnormalities in vivo. However, the cellular mechanisms controlled by MEK in primary cells remain unknown. The proposed research will investigate the effects of hyperactive Ras/MEK signaling in hematopoiesis with the following aims: (1) in the mouse model, define mechanisms by which Ras/MEK signaling increases myeloid cell production; and (2) in patient samples, test the role of MEK activity in the abnormal growth and differentiation of human MDS/MPN cells. MEK is hypothesized to stimulate increased cell division rates and/or survival in GM progenitors, as well as alter the balance of GM vs. MegE lineage commitment in immature populations such as hematopoietic stem cells. This will be addressed using time lapse cell culture methods to observe cell fate in real time, and novel single cell gene expression studies to assess lineage choice. Specific transcription factors and genes regulated by MEK will be targeted by shRNA knockdown to evaluate their functional importance in primary cells. Aim 2 will be addressed in blood and bone marrow samples from JMML and CMML patients. The platforms developed to observe murine cells will be adapted to study sorted human progenitor cells. Quantitative measurements of sensitivity to MEK inhibition in vitro will be correlated to prognostic indices from diagnostic dat. Together, these studies will reveal cellular and molecular mechanisms of aberrant hematopoiesis in MDS/MPN driven by hyperactive Ras. They will also establish systems for translating discoveries in model systems to benefit patients by improving our ability to use in vitro studies to learn how specific agents affect cell fates. Biochemical mechanisms of disease that are conserved in murine and human systems will serve as high value targets for development of novel therapies for MDS/MPN.